Energy-dependent proteolysis in reticulocytes is catalyzed by a multienzyme system for which the first step involves ATP-coupled covalent ligation of the 76 amino acid polypeptide ubiquitin to substrate proteins as the committed step for degradation of the latter. The long-term objective of the proposed research is to understand the role of ubiquitin ligation in intracellular protein degradation to provide a basis for future investigations on the relationship of this novel post-translational modification to regulation of specific enzymes, control of chromatin structure via histone ligation, and the etiology of aging. The specific aims of the proposed research are: 2) To test the hypothesis that the ATP, ubiquitin-dependent proteolytic pathway accounts for the non-lysosomal energy-dependent degradation specific for short-lived and abnormal proteins observed in all eukaryotic cells; 2) to determine the contribution of this pathway to total intracellular protein turnover; 3) to elucidate the steps involved in proteolysis by this multienzyme system; and 4) to understand the structural features of target proteins that make them susceptible to ubiquitin ligation. Antibodies specific for ubiquitin will be produced in New Zealand white rabbits and purified by affinity chromatography. The short- and long-lived pools of intracellular proteins in human lung fibroblasts (IMR-90) will be selectively labeled with 3H-leucine and 14C-leucine, respectively, then their rates of degradation compared to isotopic composition and chase of ubiquitin-protein conjugates isolated immunologically to determine which class of protein is specifically degraded by the pathway. Similar studies allowing incorporation of amino acid analogs or puromycin during 3H pulse will determine if abnormal proteins are also degraded via ubiquitin conjugates. Experiments with inhibitors of intracellular proteolysis will be used to determine the contribution of the ubiquitin pathway to total protein turnover, the steps involved in conjugate degradation, and possible participation by other energy-dependent mechanisms. Seven hepatic enzymes with different molecular properties and rates of intracellular degradation will be isolated from Sprague-Dawley rats and used as substrates for ubiquitin ligating enzymes purified from rabbit liver to provide in vitro correlations on structural features and conformational transitions involved in recognition for conjugation to corroborate the in vivo results.